Nepomuceno Diane, Kuei Chester, Dvorak Curt, Lovenberg Timothy, Liu Changlu, Bonaventure Pascal
Janssen Research and Development, LLC, San Diego, CA, United States.
Front Pharmacol. 2018 Mar 2;9:157. doi: 10.3389/fphar.2018.00157. eCollection 2018.
It is now well established that GPR139, a G-protein coupled receptor exclusively expressed in the brain and pituitary, is activated by the essential amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) via G-coupling. The affinity and potency values of L-Trp and L-Phe are within the physiological concentration ranges of L-Trp and L-Phe. A recent paper suggests that adrenocorticotropic hormone (ACTH), α and β melanocyte stimulating hormones (α-MSH and β-MSH) and derivatives α-MSH/α-MSH can also activate GPR139 . We tested this hypothesis using guanosine 5'--(3-[S]thio)-triphosphate binding (GTPγS), calcium mobilization and [H]JNJ-63533054 radioligand binding assays. In the GTPγS binding assay, α-MSH, α-MSH/α-MSH, and β-MSH had no effect on [S]GTPγS incorporation in cell membranes expressing GPR139 up to 30 μM in contrast to the concentration dependent activation produced by L-Trp, JNJ-63533054, and TC-09311 (two small molecule GPR139 agonists). ACTH slightly decreased the basal level of [S]GTPγS incorporation at 30 μM. In the GPR139 radioligand binding assay, a moderate displacement of [H]JNJ-63533054 binding by ACTH and β-MSH was observed at 30 μM (40 and 30%, respectively); α-MSH, α-MSH/α-MSH did not displace any specific binding at 30 μM. In three different host cell lines stably expressing GPR139, α-MSH, and β-MSH did not stimulate calcium mobilization in contrast to L-Trp, JNJ-63533054, and TC-09311. ACTH, α-MSH/α-MSH only weakly stimulated calcium mobilization at 30 μM (<50% of EC). We then co-transfected GPR139 with the three melanocortin (MC) receptors (MC3R, MC4R, and MC5R) to test the hypothesis that ACTH, α-MSH, and β-MSH might stimulate calcium mobilization through a MCR/GPR139 interaction. All three MC peptides stimulated calcium response in cells co-transfected with GPR139 and MC3R, MC4R, or MC5R. The MC peptides did not stimulate calcium response in cells expressing MC3R or MC5R alone consistent with the G signaling transduction pathway of these receptors. In agreement with the previously reported multiple signaling pathways of MC4R, including G transduction pathway, the MC peptides produced a calcium response in cells expressing MC4R alone. Together, our findings do not support that GPR139 is activated by ACTH, α-MSH, and β-MSH at physiologically relevant concentration but we did unravel an interaction between GPR139 and the MCRs.
现已明确，GPR139是一种仅在大脑和垂体中表达的G蛋白偶联受体，通过G偶联被必需氨基酸L - 色氨酸（L - Trp）和L - 苯丙氨酸（L - Phe）激活。L - Trp和L - Phe的亲和力和效价在L - Trp和L - Phe的生理浓度范围内。最近一篇论文表明，促肾上腺皮质激素（ACTH）、α和β黑素细胞刺激素（α - MSH和β - MSH）及其衍生物α - MSH/α - MSH也可激活GPR139。我们使用鸟苷5'-（3 - [S]硫代）-三磷酸结合（GTPγS）、钙动员和[H]JNJ - 63533054放射性配体结合试验来验证这一假设。在GTPγS结合试验中，与L - Trp、JNJ - 63533054和TC - 09311（两种小分子GPR139激动剂）产生的浓度依赖性激活相反，α - MSH、α - MSH/α - MSH和β - MSH在浓度高达30μM时对表达GPR139的细胞膜中[S]GTPγS掺入没有影响。ACTH在30μM时略微降低了[S]GTPγS掺入的基础水平。在GPR139放射性配体结合试验中，在30μM时观察到ACTH和β - MSH对[H]JNJ - 63533054结合有适度的置换作用（分别为40%和30%）；α - MSH、α - MSH/α - MSH在30μM时未置换任何特异性结合。在三种稳定表达GPR139的不同宿主细胞系中，与L - Trp、JNJ - 63533054和TC - 09311相反，α - MSH和β - MSH不刺激钙动员。ACTH、α - MSH/α - MSH在30μM时仅微弱刺激钙动员（<EC的50%）。然后我们将GPR139与三种黑皮质素（MC）受体（MC3R、MC4R和MC5R）共转染，以验证ACTH、α - MSH和β - MSH可能通过MCR/GPR139相互作用刺激钙动员这一假设。所有三种MC肽在与GPR139和MC3R、MC4R或MC5R共转染的细胞中刺激钙反应。MC肽在单独表达MC3R或MC5R的细胞中不刺激钙反应，这与这些受体的G信号转导途径一致。与先前报道的MC4R的多种信号转导途径（包括G转导途径）一致，MC肽在单独表达MC4R的细胞中产生钙反应。总之，我们的研究结果不支持在生理相关浓度下ACTH、α - MSH和β - MSH激活GPR139，但我们确实揭示了GPR139与MCR之间的相互作用。
